Assay of MCAK microtubule depolymerizing activity. During mitosis it is necessary that the duplicated chromosomes align on the mitotic spindle. Once all of the chromosomes are aligned at the metaphase plate, the sister anaphase chromatids split and migrate to their respective poles. The spindle is made up of microtubules, stiff polymers of a and B tubulin, which radiate from the centrosomes with a known polarity. Minus ends are bundled at the centrosome, whereas the plus ends are directed outward, some terminating at the centromeres of migrating mitotic chromosomes. Chromosomes can be moved with respect to the spindle by microtubule associated motor proteins. These molecules fall in two families, the Dyneins and Kinesins, which are known to have roles in vesicle transport, pigment movement, and transport of membrane-bound organelles. These two-headed motors hydrolyze ATP and utilize the energy to "walk" down microtubules, thus transporting their bound cargo. A given motor can walk eith er to the plus- or to the minus-end of microtubules. Three motor proteins have been isolated from centromeres: minus-end directed dynein, the plus-end directed kinesin CENP-E, and a poorly understood kinesin known as the Mitotic Centromere Associated Kinesin (MCAK.) MCAK was first described in 1995; however, its function still has not been determined unequivocally. It has not demonstrated an ability to actually move along microtubules but still appears to be important in chromosomal segregation. Preliminary studies suggest that the Xenopus homolog of MCAK, called XCCMI has a microtubule depolymerizing activity, possibly effecting chromosomal movement by modulating microtubule stability. We set out to assay MCAK for this activity. Stable fluorescent GMPCPP microtubules were polymerized and pipetted into a silanized flow cell. The flow cell was then washed with a lmg/ml solution of casein in order to remove unbound tubules and to coat glass surfaces. Finally, 0.51 M MCAK was flowed in (or heat-killed MCAK negative control.) Microtubules were then visualized by fluorescence microscopy, and a given set of microtubules was videotaped for three seconds every ten minutes, to enable assessment of length change. Experimental and control groups displayed microtubules shortening 0.93+0.09 microns/hr and 0.81+0.13 microns/hr respectively, not statistically significantly different. We are now assaying for ability to depolymerize free-floating tubules. Determining how such molecular motors work will also help understanding how cells achieve morphology, move, and exert forces on their environments.